Abstract-Summary Liver disease claims over 38,000 lives every year in the U.S. alone. Unfortunately, there are no dialysis or left ventricular assist device equivalents for liver failure and a liver transplant is the only treatment. Yet thousands of potentially transplantable livers are discarded every year due to limitations in organ preservation times, which are on the order of hours. The broad objective of this program is the development of a comprehensive system for the long-term banking of livers for transplantation. This system will dramatically extend preservation times for human livers, thus increasing the availability and efficient use of transplantable donor livers. Our approach combines the best strategies for cryopreserving tissues in a vitreous ?glassy? or ?amorphous? state with a novel, breakthrough nanowarming technology, which utilizes infused radiofrequency-excited biocompatible nanoparticles to rewarm tissue. Unique to this technology, nanowarming can rewarm large tissue volumes rapidly and uniformly, which mitigates one of the major challenges in scaling cryopreservation technology to whole organs: ice growth and fracturing/cracking of tissues during the critical rewarming phases. In this project, we will integrate recent advances in machine perfusion with optimized loading/unloading procedures, next generation non-toxic cryopreservation solutions, innovative breakthrough nanowarming technologies using biocompatible nanoparticles, and powerful tissue imaging methods (e.g. cryomacroscopy, uCT, MRI) to enable a convergence of state-of-the-art technologies to develop a comprehensive preservation protocol that will maximize tissue viability. In Aims 1 and 2, we use representative precision cut liver slices (up to 50mls volume) to select the best available vitrification cocktail specific for liver tissue and to determine the critical cooling and critical warming rates required to achieve ice-free and stress-free cooling and warming. In Aim 3, a comprehensive multi-thermic machine perfusion (MTMP) protocol will be applied to the whole rat liver to validate tolerance to the addition and removal of the vitrification cocktail (from Aim 1) containing nanoparticles. Finally, whole rat livers will be vitrified using the comprehensive MTMP protocol determined in Aims 1-3 and rewarmed via nanowarming to show feasibility for successful vitrification and nanowarming without damaging ice crystal formation or tissue fracturing. Vitrified whole livers rewarmed using traditional convective methods will serve as a control group for comparison. Altogether, this constitutes an important standalone project whose sub- strategies are generally applicable and scalable to improve the long-term preservation of cells, tissues, and whole organs. The subsequent Phase II study will target optimization of the MTMP/nanowarming protocol for the preservation of transplant quality liver function and scale up to human livers. Importantly, success of these novel sub-strategies (MTMP, nanowarming, etc.) individually or in combination will likely enable further breakthroughs in biopreservation, tissue engineering, bioemedical research and clinical practice.